Folic Acid Preconditioning Alleviated Radiation-Induced Ovarian Dysfunction in Female Mice

Radiological therapy/examination is the primary source of artificial radiation exposure in humans. While its application has contributed to major advances in disease diagnosis and treatment, ionizing radiation exposure is associated with ovarian damage. The use of natural products, either alone or as an adjunct, has become increasingly common for reducing the side effects of radiological therapy during disease treatment. Herein, we explored the protective effect of folic acid (FA), a widely used B vitamin, against radiation-induced ovarian injury and its mechanism of action. Female mice with normal ovarian function were randomly divided into control, FA, radiation, and radiation + FA groups. The intervention strategy included daily intragastric administration of FA (5 mg/kg) for 3 weeks prior to radiation exposure. Mice in the radiation and radiation + FA groups received a single dose of 5 Gy X-ray irradiation. Changes in the estrous cycle were then recorded, and ovarian tissues were collected. Pathophysiological changes as well as reproductive and endocrine-related indexes were determined via H&E staining, immunohistochemistry, Western blot, and ELISA. The reproductive performance and emotional symptoms of animals were also monitored. Our results indicated that FA intervention effectively alleviated ovarian damage, leading to more regular estrous cycles, lesser impairment of follicular morphology and endocrine status, as well as greater germ cell preservation. Reduced levels of oxidative stress, inflammation, and enhanced DNA repair were associated these changes. FA pre-administration improved the reproductive performance, leading to higher pregnancy rates and greater litter sizes. Further, the anxiety levels of animals were significantly reduced. Our results indicate that FA pre-administration significantly alleviates radiation-induced ovarian damage in rodents, highlighting its potential as a protective strategy against radiation exposure in the female population.

INTRODUCTION
Radiation is distributed throughout the environment, emanating from natural sources as well as anthropogenic activity. Cosmic rays constitute the main source of radiation exposure for humans in the natural environment, while medical radiation is the major source of artificial radiation (1,2). Medical ionizing radiation (IR) is widely used for clinical diagnosis and cancer therapy. Thus, radiation exposure is generally unavoidable and is predicted to induce health hazards, with the ovaries being among the organs most susceptible to radiation-induced injury (3). Therefore, increasing attention has been directed toward minimizing ovarian radiotoxicity.
Radiotherapy represents a major strategy for the treatment of cervical cancer, endometrial cancer, rectal cancer, and other malignancies (4)(5)(6). Diagnostic applications of radiation, such as mammography, are the gold standard for diagnosing various diseases, such as breast cancer (7). While the application of radiation in diagnosis and treatment has improved disease management, radiation exposure has also been shown to have deleterious effects on various living organisms, including humans, with organs of the reproductive system being the most susceptible to radiation damage (8)(9)(10)(11).
Ovarian germ cells are highly sensitive to radiation, with exposure, such as through radiotherapy, causing inflammation and oxidative stress, which in turn leads to germ cell apoptosis (12,13). Ovarian follicles composed of germ cells and granulosa cells represent the basic functional units of the ovary (14). Radiation-induced reduction of the follicle reserve and follicle atresia adversely affect reproductive function and hormone secretion, compromising female fertility, with various accompanying symptoms that severely impact the physical and mental health of women. Radiation exposure-induced premature ovarian failure (POF)-like features have been widely reported and are receiving increasing attention (15,16). Moreover, medical radiation poses potentially serious risks to the fertility of female patients as well as female medical practitioners. At present, strategies for mitigating radiation-induced ovarian damage mainly include hormonal regulation, cryopreservation of germ cells and ovarian tissues, assisted reproductive technology, stem cell transplantation, and pharmacological therapy. However, these are associated with various disadvantages, including side effects, high cost, poor compliance, and suboptimal efficacy (17,18). Taken together, the prevention and restoration of ovarian function following radiation-induced damage represents a major challenge in the clinic.
Folic acid (FA) is a B vitamin that serves as a coenzyme in nucleic acid and amino acid metabolism, thus participating in a number of important biochemical reactions within cells. Mounting evidence has shown that FA has antioxidant and antiinflammatory properties (19,20). Further, it is widely utilized for improving female fertility in the clinic (21). The therapeutic effects of FA overlap with the adverse effects of radiation-induced ovarian damage (22). It has been established that compounds with antioxidative properties harbor radioprotective capacity (23,24). However, whether FA could protect against radiationinduced ovarian damage remains unclear. The pharmacological properties of FA and the pathogenesis of radiation-induced injury suggest that the former has potential for alleviating radiation-induced ovarian injury. In the present study, we aimed to investigate whether prophylactic FA administration could alleviate ovarian irradiation-induced reproductive and endocrine-related dysfunction in female mice.

Animal Model and Treatment
Ten-week-old female C57BL/6 mice were obtained from Beijing Vitong Lihua Co., Ltd. The mice were kept in a controlled standard environment (23±1 • C, 12-h light/dark cycles), with free access to standard food and clean water. After basic physical examination and confirmation of regular estrous cycles (based on morphological changes in vaginal cytology smears obtained every morning at 8:30 am and observed under a microscope), female mice were randomly assigned to one of four groups: control group (Con), FA pre-administration control group (FA), radiation exposure group (RA), and FA pretreatment + radiation exposure group (RA+FA). FA pre-administration dosage and protocol were selected on the basis of our previous study (25). Briefly, FA (5 mg/kg; Sigma, St. Louis, MO, USA) was administered daily via gavage. Radiation exposure was performed after 3 weeks of FA pre-administration. Prior to radiation exposure, animals were anesthetized with 1% pentobarbital sodium (40 mg/kg). Only the ovarian parts of the mouse abdomen were fully exposed, while the rest was covered with lead plates. Local radiation exposure was carried out using X-rays from an Elekta Synergy linear accelerator (Elekta AB, Sweden) at a distance of 90 cm, and a single dose of 5 Gy (absorbed dose rate: 1.5 Gy/min) was administered (26). All animal experimental procedures were formally reviewed, approved, and supervised by the ethics committee of the Guangdong Women and Children Hospital (202101345). All efforts were made to minimize animal suffering.

Ovarian Morphology Observation
At the end of experiments, mice were euthanized by cervical dislocation, and ovarian tissues were rapidly extracted. For hematoxylin and eosin (H&E) staining, tissues were fixed in 4% paraformaldehyde fixative solution for 24 h. After routine gradient dehydration and paraffin embedding, ovarian tissue samples were cut into 5-µm-thick sections and stained with H&E and terminal deoxynucleotidyl transferase dUTP-mediated nickend labeling (TUNEL) staining (C1090, Biyuntian, China). A microscope (Olympus DP71, Tokyo, Japan) was used to capture images of the sections, and follicles were then quantified using CaseViewer software (3D Histech Ltd., Budapest, Hungary).

Immunohistochemistry
The ovarian tissues were cut into 5-µm-thick sections. After antigen retrieval, the ovarian sections were incubated overnight with a rabbit polyclonal antibody against AMH (ab103233, 1:2000, Abcam). After washing, the immunoperoxidase-labeled Donkey Anti-Rabbit IgG secondary antibody (1:2000, LS-C60950, LSBio) was applied to all slides at room temperature for 1 h. After washing with PBS three times, DBA was used for color rendering, and the nuclei were re-stained with hematoxylin for 2 min. Finally, the slides were sealed with neutral adhesive for observation, and the AMH signals were acquired using a light microscope (BX51, Olympus, Tokyo, Japan).

Open Field Test
The space exploration ability and anxiety-like behavior of mice were evaluated in the open field test. The mice were acclimated to the laboratory environment for 30 min prior to the experiment. As previously described, the mice were gently placed at the center of an open box and allowed to explore the environment freely for 5 min. The spontaneous activity of mice within 5 min was recorded and evaluated using activity analysis software (Ethovision 7.0, Noldus, Wageningen, Netherlands). Acquired data included the total traveled distance and the stay time of mice in the central grid. After testing each mouse, the experimental device site was wiped with 75% alcohol to avoid odor interference in all behavioral experiments.

Elevated Plus Maze Test
Anxiety-like behavior was examined using the elevated plus maze as previously described (27)

Statistical Analysis
GraphPad PRISM version 6 (GraphPad Software, Inc, La Jolla, CA, USA.) was used for statistical analysis and illustration. All data were expressed as the mean ± standard error of mean (SEM). One-way analysis of variance (ANOVA) was used to assess the differences between multiple groups, followed by a Bonferroni's post hoc test. A P < 0.05 indicated statistical significance.

Protective Effects of FA Pre-administration on Radiation-Induced Menstrual Cycle Disturbance and Ovarian Atrophy
The estrous cycle was evaluated based on vaginal smear slides stained with crystal violet immediately after RA injury. In terms of regularity, mice in the Con and FA groups had a similar regular menstrual cycle. However, irregular menstrual cycles were observed in the RA group; this could be reversed by FA pre-administration which improved cycle regularity ( Figure 1A). The ovarian weight was similar between the Con and FA groups. However, the ovarian weight of mice in the RA group was significantly decreased (Con: 0.006775 ± 0.00043 g, FA: 0.006925 ± 0.0002644 g, RA: 0.003475 ± 0.0004113 g, RA+FA: 0.005388 ± 0.0003303 g). Thus, FA pretreatment significantly prevented ovarian atrophy compared to radiation alone ( Figure 1B).

FA Pre-administration Alleviates Radiation-Induced Ovarian Follicle Damage
We then assessed morphological changes in ovarian tissue via H&E staining which revealed that ovarian tissues of the Con and FA groups exhibited normal morphology without pathological changes (Figure 2A). The structure of ovarian tissues in the Con and FA groups was normal, and the follicles at various stages of development as well as corpus lutei were abundant. After radiation, the ovaries exhibited atrophy. In the RA group, the number of follicles at nearly all stages were decreased, and the number of atretic follicles was increased, accompanied by fibrosis in the stroma. Compared with the RA group, the FA + RA group exhibited obvious improvement, that is, the total number of female follicles increased, a large number of primordial, primary, secondary, and antral follicles were preserved, with fewer atretic follicles present ( Figure 2B). Thus, FA pre-conditioning alleviated ovarian follicle damage after radiation exposure.

Effect of FA Pre-administration on Radiation-Induced Reproductive Hormone Dysregulation
Since the ovaries are responsible for the synthesis and secretion of hormones that regulate reproductive function, we examined the levels of serum sex hormone markers and ovarian reserve markers after radiation. Compared with the Con group, the serum levels of FSH and LH were markedly increased upon radiation, with FA pre-administration lowering both (Figures 3A,B). Moreover, the concentrations of P, E2, and AMH in the RA group were lower than those in the Con group. These hormones were upregulated in the RA + FA group relative to the RA group (Figures 3C-E). Similarly, AMH immunohistochemistry staining in ovarian tissue indicated that irradiation decreased the AMH-positive area relative to the Con group ( Figure 3F). Conversely, FA pre-administration upregulated AMH signal density relative to the RA group.

Protective Effect of FA Pre-administration Against Radiation-Induced Oxidative Stress and Inflammatory Injury of Ovarian Tissue
To evaluate the protective mechanism of FA on ovarian viability, TUNEL staining was used to assess ovarian apoptosis. Increased ovarian follicle apoptosis was observed after radiation injury, while FA pre-administration effectively decreased the number of TUNEL-positive cells in the ovarian tissue (Figures 4A,B). The levels of factors related to oxidative stress (SOD, MDA, GSH-Px, and ROS) in ovarian tissue homogenates were analyzed using ELISA or western blot. As shown in Figures 4C-F, radiation downregulated the activity of SOD and GSH-Px while upregulating MDA and ROS levels. Compared with the RA group, FA pre-administration significantly upregulated SOD and GSH-Px, while downregulating MDA and ROS levels. Nrf2/HO-1 is an intracellular system driving the antioxidant response. HO-1 and NRF2 levels were significantly lower in the RA group than in Con group. Further, the expression of NRF2 and HO-1 was effectively maintained in the RA+FA group following irradiation (Figures 4G,H). Western blot results further confirmed that radiation injury dramatically upregulated the oxidative stress response as well as inflammatory cytokines, TNF-α, IL-1β, and IL-6, while FA pre-administration alleviated these effects (Figures 4G,H).

Molecular Mechanism Underlying the Protective Effect of FA Pre-administration Against Radiation-Induced Ovarian Injury
Given the importance of germ cells for normal ovarian function, we examined the effects of radiation exposure on FIGURE 2 | Effect of folic acid (FA) pre-administration on ovarian pathological damage after radiation injury. (A) The results of hematoxylin and eosin (H&E) staining indicated that FA pre-administration relieved ovarian histopathological damage. Red arrow: primordial follicles; yellow arrow: primary follicles; green arrow: secondary follicles; blue arrow: antral follicles; black arrow: atretic follicles. Scale bar, 400 µm. (B) Relative quantification of the ovarian follicles for each group. There was no significant difference in follicle number between the Con group and FA group (p > 0.05). The numbers of primordial follicles, primary follicles, secondary follicles, and antral follicles in the RA group were significantly lower than those in the Con group. FA pre-administration significantly relieved the effect of radiation on the ovarian tissue structure and increased the number of ovarian follicles, thus enhancing ovarian reserve capacity. Con: control group, FA, FA pre-administration control group; RA, radiation exposure group; RA+FA, FA pretreatment + radiation exposure group. Error bars, standard error of mean (SEM). N = 7, *p < 0.05, **p < 0.01, and ***p < 0.001. germ cells within ovarian tissues. As shown in Figure 5A, the germ cell marker mouse vasa homolog (MVH) was visualized via immunofluorescence staining. Radiation exposure induced a marked decrease in MVH-positive cells, while FA pre-administration preserved germ cells relative to the RA group. These results were confirmed via quantitative immunoblot analysis (Figures 5B,C). As ovarian germline stem cells are radiosensitive, we assessed the effect of FA on the expression of Oct4, a major transcription factor for ovarian stem cell self-renewal. Oct4 expression was reduced in mice with radiation-induced ovarian injury, which was reversed via FA pre-administration ( Figure 5D). Immunoblot analysis of Ki67 and PCNA further demonstrated that FA pre-administration attenuated radiation-induced suppression of ovarian cell proliferation (Figures 5B,E,F). Accordingly, we hypothesized that the recovery of ovarian cell proliferation under FA pre-administration was due to enhanced DNA repair. To assess this, we examined the expression of DNA repairassociated proteins. Western blot analysis indicated that FA pre-administration markedly upregulated the protein expression of DNA repair markers ATM and RAD51 upon irradiation (Figures 5B,G,H). Taken together, these results indicated that FA pre-administration reverses the radiation-induced negative effect on female mouse reproductive ability by enhancing DNA repair capacity and thus improving germ cell radioresistance.

FA Pre-administration Alleviates Radiation-Induced Reproductive Dysfunction in Female Mice
Our results prompted us to further determine whether FA preadministration has a beneficial effect on animal reproductive performance. Male mice with normal fertility were mated with each group of females. The pregnancy rate among mice in the RA group was significantly lower than that in the Con and FA groups Each ELISA assay was repeated three times. (G,H) Western blot was performed to detect the protein levels of HO-1, Nrf2, TNF-α, IL-1β, IL-6, and Cleaved caspase-3 in ovarian tissues from each group.in ovarian tissues from each group. HO-1, heme oxygenase 1; Nrf2, nuclear factor-erythroid 2-related factor 2. TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; IL-6, interleukin-6; Con, control group; FA, FA pre-administration control group; RA, radiation exposure group; RA+FA, FA pretreatment + radiation exposure group. Error bars, standard error of mean (SEM). N = 8, *p < 0.05, **p < 0.01, and ***p < 0.001. (both over 80%) (Figure 6A), with more than 66% of female mice in the RA group experiencing unsuccessful pregnancies. The pregnancy rate among female mice pre-administered FA was about 66%. In addition, the litter size of mice in the RA group was significantly reduced, but this effect was effectively reversed via FA pre-administration (Figures 6B,C).

FA Pre-administration Relieves Ovarian Irradiation-Induced Emotional Disturbance
Patients undergoing ovarian radiation are known to experience emotional symptoms (28). Therefore, we examined the emotional behaviors in each group. During the open field test (Figure 7A), there were no significant differences in the distance traveled between groups (Figure 7B). However, radiation-exposed mice exhibited a significantly shorter time in the center area of the arena. FA pre-administration increased center area exploration relative to that of RA group mice ( Figure 7C). The lower center activity indicated an increased level of anxiety among RA group animals. Assessment of anxiety-like behavior with an elevated plus maze was then carried out. We observed that radiationexposed mice spent less time exploring the open arm than Con mice (Figures 7D-F), suggesting increased anxiety-like behavior in the former. In contrast, FA pre-administration significantly increased open-arm entries and exploration time, suggesting an anxiolytic effect in mice that underwent ovarian irradiation.

DISCUSSION
Considering the increasing incidence of gynecological and gastrointestinal tumors, a greater number of female patients undergo radiotherapy, which is a well-established treatment approach for various malignancies. While these female patients experience significantly improved treatment outcomes and life expectancy, they are under an increased risk of radiation damage to adjacent organs, with the ovaries being among the most common sites of radiation-induced injury. Women who suffer from ovarian radiation damage may develop secondary conditions such as amenorrhea, infertility, and premature ovarian failure, which severely affects their physical and mental health. Thus, minimizing the side effects of radiation therapy remains an unsolved clinical challenge. China has the largest female population in the world, which implies that a great number of Chinese women suffering potential risks for radiationrelated ovarian damage, highlighting the urgent need for effective protection methods against radiation.
Radiation, such as in the form of X-ray irradiation, exhibits strong tissue penetration (29). Although physical protection and radiation dosage control are effective strategies to reduce radiation damage, they have certain limitations regarding operability, maneuverability, objectivity, and repeatability. Therefore, the focus should shift toward strategies based on drug intervention, while considering the adverse effects of potential therapeutics. FA is a physiologically essential vitamin and has been found to reduce side effects due to treatment in multiple organs (30,31). Herein, we report for the first time the protective effect of FA pre-administration on radiation-induced ovarian injury. We demonstrated that FA pre-administration protected female mice against radiation-induced endocrine and reproductive dysfunction.
Clinical studies have identified radiation exposure as a major risk factor for premature ovarian failure (32). It has a strong detrimental effect on DNA synthesis (33). Further, radiation induces oxidative stress, inflammation, and the subsequent apoptosis of germ cells. Normal ovarian function depends on the continuous growth and development of follicles. Radiotherapy not only kills proliferating tumor cells, but also leads to the apoptosis of germ cells, thus causing follicle failure. Previous studies have demonstrated that FA has antioxidant and antiinflammatory effects, suggesting that it may hold potential for the mitigation of radiation damage in the ovaries (23,24). However, the effect of FA on radiation damage has remained unclear. Therefore, in this study, we established a mouse ovarian radiation injury model and explored the protective effect of FA pre-administration against radiation-induced ovarian injury. We first examined apparent disturbance of the estrous cycle following ovarian injury. Animals exhibited irregular estrous cycles following a single moderate dose of X-ray radiation, which further confirmed damage to the female reproductive system caused by ionizing radiation. This is consistent with previously reported ovarian dysfunction symptoms of menstrual irregularity (34). Animals in the FA pre-administration group had more regular menstrual cycles, suggestive of a protective effect of FA on ovarian function. This supports our hypothesis that FA preadministration may be a potential strategy for mitigating ovarian radiation damage.
Pathological and morphological observation results revealed atrophy of ovarian tissue and a significant reduction in the number of follicles following radiation exposure, which was consistent with the findings of previous reports (12,13). In this study, we showed that FA-pretreated animals exhibited significantly lower ovarian tissue atrophy, more follicles were reserved, and there was a reduced occurrence of atresia. The reserved follicle is the basic functional unit of the ovary for reproduction and endocrine regulation. Further, the existence of oocytes and granulosa cells in follicles is the physiological basis of reproductive and endocrine follicular function (35,36). Follicular atresia, which is observed during ovarian injury, is the result of ovarian granulosa cell and oocyte apoptosis, eventually leading to symptoms of premature ovarian failure. Our experimental results indicated that, compared with mice of the radiation group, those receiving FA pre-administration had significantly improved serum reproductive hormone levels and better ovarian endocrine function, suggesting that FA could alleviate the follicular damage caused by radiation. Correspondingly, FA pre-administration remarkly reduced the number of TUNELpositive apoptotic cells within follicles. Oxidative stress is one of the main mechanisms of radiation injury and leads to cell apoptosis (37). Nrf2 is the most active among leucine zipper transcription factor, driving the downstream expression of genes that encode antioxidant proteins to facilitate the body's defense against oxidative stress and to protect cells against radiation (38). The expression of HO-1 has been widely demonstrated to have anti-inflammatory and antioxidant effects (39). FA preadministration upregulated the expression of various antioxidant response-related molecules, including HO-1, in the ovaries and significantly suppressed oxidative stress and inflammation levels caused by radiation exposure, thus playing a role in alleviating radiation-induced damage. This is consistent with the previously reported effect of FA on spleen injury induced by lead acetate and cardiac dysfunction induced by hyperhomocysteinemia (40,41).
The suppression of oxidative stress and inflammation contributes to the preservation of functional cells within the ovaries. Immunohistochemistry analysis confirmed that FA pre-administration resulted in a significant increase in preserved reproductive cells within the ovaries. In view of the effects of FA on DNA synthesis and repair, we explored whether pre-administration could promote DNA repair (42). ATM and its downstream factor RAD51 are key molecules involved in DNA repair (43). We found that FA pre-administration upregulated the expression of both ATM and RAD51. This provided a basis for the specific mechanism underlying the protective effect of FA in damaged follicles. Taken together, the current findings support the potential of FA in mitigating reproductive impairment in animals.
We examined the actual reproductive performance of animals using two main indicators, namely pregnancy rate and litter size, which reflect ovarian and follicular function at two levels. Most female animals in the control and FA groups became pregnant within four weeks of co-caging with males of normal reproductive function. Pregnancy rates were significantly reduced in the animals exposed to radiation. We suspect that the ovarian impairment-induced POF-like hormonal state suppresses the reproductive function of animals, reducing their libido and leading to lower reproductive performance. Litter size, on the other hand, is an indicator of the number of functional follicles within the ovary. Damage to the ovarian follicles reduces their ability to bind to sperm. The observed litter sizes were consistent with the follicular morphology, confirming the protective effect of FA preadministration against radiation-induced impairment of ovarian reproductive function.
Radiation exposure-associated emotional disturbances are common and manifest as anxiety and depression, among other symptoms (44,45). Therefore, we examined emotion-related symptoms in the present study. The results indicated that female mice subjected to ovary irradiation exhibited significantly increased levels of anxiety. These observations were similar to the symptoms observed in patients with POF (46). Previous studies have shown that radiation can cause mood changes in animals (44). In the present study, we covered other parts of the body to exclude damage to organs other than the ovaries. Thus, we report for the first time the direct relationship between radiation damage to the ovaries and mood change. A previous report demonstrated that FA can regulate emotions in women (25). We further characterized the regulatory role of FA in radiation-induced mood abnormalities. Our findings suggested that FA significantly alleviated the increased anxiety caused by ovarian radiation exposure, which may be related to the improvement of peripheral hormone levels promoted via FA preadministration.
Our study fully considered and implemented the 3R principle in animal studies, and, although we have preliminary evidence for the protective effect of FA against radiation-induced ovarian damage, several important questions remain to be answered. First, the optimal combination and dose for intervention strategies remain to be determined. As a measure of reproductive function, the pregnancy rate indicates the percentage of females with bulging bellies who give birth to pups. Related factors, such as abortion, were not considered and studied in this process, which must be addressed in future studies. In addition, we did not examine the later development of progeny. Although we observed the activation of DNA repair mechanisms in the ovary, the protective effect of FA on radiation teratogenesis remains to be explored.

CONCLUSIONS
In summary, the results of the present study further enhance our understanding of radiation-induced ovarian damage pathogenesis and progression. The present study demonstrated that FA pre-administration can effectively alleviate the ovarian damage induced by radiation in female mice, restore ovarian function, and relieve symptoms such as premature ovarian failure. Taken together, we offer a novel potential strategy for preserving ovarian function in female populations with potential radiation exposure risks.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The animal study was reviewed and approved by Ethics Committee of Guangdong Women and Children Hospital.


s2fieldsofstudy: Biology Medicine

text:
Proposed Cellular Function of the Human FAM111B Protein and Dysregulation in Fibrosis and Cancer

FAM111B gene mutations are associated with a hereditary fibrosing poikiloderma known to cause poikiloderma, tendon contracture, myopathy, and pulmonary fibrosis (POIKTMP). In addition, the overexpression of FAM111B has been associated with cancer progression and poor prognosis. This review inferred the molecular function of this gene’s protein product and mutational dysfunction in fibrosis and cancer based on recent findings from studies on this gene. In conclusion, FAM111B represents an uncharacterized protease involved in DNA repair, cell cycle regulation, and apoptosis. The dysregulation of this protein ultimately leads to fibrotic diseases like POIKTMP and cancers via the disruption of these cellular processes by the mutation of the FAM111B gene. Hence, it should be studied in the context of these diseases as a possible therapeutic target.

INTRODUCTION
Mutations of the human FAM111B gene are also associated with a rare multisystemic fibrosing disease-poikiloderma, tendon contracture, myopathy, and pulmonary fibrosis (POIKTMP, which is the adopted terminology for this disease) (1)(2)(3)(4). FAM111B gene mutations are also implicated in other clinical manifestations such as progressive osseous heteroplasia (POH) (5), autism spectrum disorders (6), modification of genes associated with cognitive development (7), nevus of Ota with choroidal melanoma (8), and mutations of unknown clinical significance/common genetic polymorphism (9). Furthermore, FAM111B gene mutations correlate positively with increased cancer predisposition (10)(11)(12). Moreover, the overexpression of this gene in cancer cells is associated with increased cancer progression and poor clinical outcomes (13)(14)(15)(16). Although there are about 19 reported FAM111B mutations (Supplementary Table 1) to date (3-5, 10, 12, 17-24), the physiological function(s) of the FAM111B protein and its dysfunctional role in diseases like fibrosis and cancers are not well established. This review highlighted the recent proposed cellular functions of the FAM111B protein to provide insights into the molecular basis of FAM111B protein dysregulation in POIKTMP and cancers.

FAM111B AS A PROTEASE
Although the FAM111B gene products, specifically the protein, are not well characterized, various bioinformatics studies have predicted the presence of a putative trypsin/cysteine proteaselike domain at the C-terminus of the FAM111B protein (2,14,25). The functional importance of this trypsin-like protease domain was demonstrated by Kawasaki et al. (14). A FAM111B protease domain-deletion construct failed to reduce cell proliferation in a FAM111B gene knockout cell line.
FAM111B is a paralog of FAM111A, the first member of this family of proteins, which also has this trypsin/cysteine-like protease domain (25,26). FAM111A is a reported DNAbinding protein and plays an essential role in mitigating protein obstacles on replication forks by cleaving DNA-protein crosslinks (DPCs) (26,27). The interaction of FAM111B with calpains (calcium-dependent cysteine proteases), specifically calpain small subunit 1 (CAPNS1) and calpain 1 (CAPN1), may also suggest the proteolytic function of FAM111B (12). FAM111B also shares about 45% sequence homology with FAM111A protein. However, both proteins' three-dimensional (3D) structures are unavailable. Structural homology modeling using the E. coli DegS protease as a template has indicated shared structural homology most strikingly at the predicted catalytic domain (25), implying further the function of FAM111B as a protease.

FAM111B IN THE CELL CYCLE
One of the first published data supporting FAM111 B's involvement in the cell cycle was from a multi-omics gene study, which showed the steady increase in FAM111B transcript levels during the G1 phase leading to detectable protein accumulation by the S phase (28). These results were further supported in a FAM111B-deleted lung adenocarcinoma (LUAD) cell line. A significant reduction occurred at the S and G2/M phases, with more cells remaining in the G 0 /G1 phases (14). This study suggested further that FAM111B degrades p16 (CDKN2A), a tumor-suppressor protein of the two classic cell cycle kinases: cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) (29). These kinases form a cyclin D1-CDK4/6 complex which phosphorylates the retinoblastoma protein (pRb1) (14,30). The phosphorylated pRb1 dissociates from E2F, enabling cell cycle progression from the G1 to S phase (31). Furthermore, a marked increase of p16 and phosphorylated pRb1 and E2F expression levels was reported in a FAM111B knockout cell line, leading to these cells' lack of progression to S and G2/M and cell cycle arrest at the G 0 /G1 phase (14). Insights into the role of FAM111B protein during the S phase of the cell cycle were also provided by discovering the interaction between FAM111B and DNA-binding proteins like RFC1 (replication factor C subunit 1) and PCNA (proliferating cell nuclear antigen) (25). The RFC is a five-subunit protein that assists with loading PCNA into DNA, a crucial step in DNA replication (32). The disruption of these replication forks complex proteins: PCDNA and RFC were reported in FAM111B-dysregulated (overexpressed) and patient-derived mutant-expressing cells (25). Another study reported a strong association of FAM111B with G2/M phase proteins, precisely cyclin B1 (CCNB1), and the dual-specificity protein phosphatase CDC25C (13). For a successful G2/M transition, cyclin B must form a complex with CDK1 (a.k.a. CDC2 or p34). This complex is kept inactive by phosphorylation of CDK1 and activated by dephosphorylation CDC25C (33,34). CDC25C also plays a crucial role in the cell cycle as a checkpoint protein, especially in DNA damage, thus ensuring accurate genomic information to daughter cells (35). Hence, a positive correlation of FAM111B with CCNB1 and CDC25C suggests that FAM111B promotes cell cycle progression into the mitotic phase. Moreover, mutations in the FAM111B gene were also shown to cause replication and transcriptional shutdown, disruption of microtubule network integrity at the M phase, and increased apoptosis (25). Hence, the proposed functions of FAM111B in cell cycle and division are hereby summarized in Figure 1A.

FAM111B IN DNA REPAIR AND APOPTOSIS
Mutations in the FAM111B gene are associated with the loss of genome stability and integrity, suggesting its role in DNA repair and genome integrity (12,36). The exact details of how FAM111B corrects the DNA are yet to be determined. However, given the possible functional overlap between FAM111A and FAM111B, we can hypothesize the possible mechanism of repair based on the known functions of FAM111A. The FAM111A, as a DNA-protein crosslink (DPC) protease, plays a role in the removal of DPCs that stall DNA replication (26,36). DPCs occur when proteins within the cell become covalently trapped on a DNA strand due to exposure to endogenous/exogenous (e.g., formaldehyde) and chemotherapeutic agents. These DPCs interfere with DNA replication, repair, and transcription, eventually leading to genomic instability in the form of DNA double-strand breaks (36). FAM111A, among other DPCs, proteases, and possibly FAM111B are involved in the proteolytic cleavage of DPCs, hence forestalling DNA damage, genome instability, and DNAdamage-mediated apoptosis (25,26,36). Following the repair of DPCs, FAM111B and FAM111A should possibly undergo autocleavage like the essential mammalian DPC protease, SprTlike N-terminal domain (SPRTN) (37,38). Although not well characterized, this regulatory process is thought to ensure the nonspecific degradation or disruption of protein complexes crucial for maintaining genome integrity, such as the chromatin-associated RNA polymerase II (RPB1), PCDNA, or other histone proteins (25,36,39). Its interaction with CAPNS1 further suggests the role of FAM111B in DNA repair-CAPN1 [calpain small subunit 1 (Calpain 1)], USP1 (ubiquitin-specific protease 1), and FANCD2 (Fanconi anemia group D2 protein) (12). FAM111B interacts with CAPNS1, which forms a complex with CAPN1 (i.e., CAPNS1-CAP1) that stabilizes USP1 deubiquitinase, which, in turn, modifies FANCD2, an essential protein involved in various DNA damage response pathways (12,40). Furthermore, the downregulation of antiapoptotic genes such as BCL-2 (B-cell lymphoma 2) and BAG3 (Bcl-2-associated athanogene 3) has been reported in FAM111B knockout cells (13,25). Since there is evidence suggesting that FAM111B is involved in the DNA repair pathway and apoptosis (13,25,36), this finding begs the possible involvement of other DNA-damage-induced stress response pathways associated with FAM111B. The answer to the question was hinted at by Sun et al. (2020) by linking FAM111B to the p53 signaling pathway using KEGG enrichment analysis (13). The phosphorylation and activation of the p53 pathway are known to cause cell cycle arrest, cell senescence, or apoptosis in response to intracellular stresses such as DNA damage (41,42). The FAM111B gene is reportedly enriched in the p53 signaling pathway, and the knockout of FAM111B reportedly affected the expression of p53-related genes such as BAG3, BCL-2, and CCNB1 (13). Thus, it is firmly possible that FAM111B is part of the p53 signaling pathway. Interestingly, previous studies have indicated that the knockout of FAM111B does not affect the p53 gene or protein expression (13,14), suggesting that FAM111B operates downstream of p53. Hence, we propose that FAM111 B's function in DNA repair and apoptosis is mediated by the direct or indirect downstream stream activation of FAM111B by the p53 (Figure 1B).

FAM111B IN FIBROSIS: HEREDITARY FIBROSING POIKILODERMA (POIKTMP)
FAM111B gene mutations are associated with POIKTMP, a hereditary multisystemic fibrosis disorder (2). This syndrome is characterized by fibrosis in multiple organs such as the skin and lungs (1, 2); however, the molecular basis of the FAM111B mutations in this disease remains unclear. Fibrosis results from excessive extracellular matrix component (ECM) deposition by activated fibroblasts when trigged by multiple stimuli (43). One such trigger is chronic from a sustained release of proinflammatory cytokines such as TGFb and IL-13 (44). FAM111B and FAM111A gene mutations are suggested to result in the rapid cleavage of both proteins, possibly due to mutational gain of function of their protease activity (10). This rapid selfcleaving activity of these proteins, specifically FAM111B in POIKTMP, could result in the ineffective removal of DPCs or the disruption/degradation of PCNA-associated proteins like RFC1 and chromatin-bound RPB1 (36, 39), thus leading to failure in DNA repair and, ultimately, genome instability.
Furthermore, incomplete DNA repair and genome instability could result in excessive cell cycle arrest (given the regulatory role of FAM111B on p16) and ultimately apoptosis which may, in turn, trigger chronic inflammation due to the incomplete clearance of apoptotic cells (efferocytosis) by granulocytes (45), hence the chronic inflammation seen in the various tissues affected by POIKTMP. Furthermore, chronic inflammation is known to cause several types of alopecia, such as primary scarring alopecia (PSA) (12,46), and may also explain the alopecia seen in patients with POIKTMP. Interestingly, mutations in DNA repair genes, genomic instability, and chronic inflammation play a significant role in other hereditary disorders like Hutchinson-Gilford Progeria Syndrome (36, 46,47); Progeria causes clinical manifestations like POIKTMP as alopecia and joint contractures (46). This information further supports the view that genomic instability from FAM111B gene mutations causes POIKTMP (15,36). However, the molecular mechanisms behind how FAM111B gene mutations lead to these diseases remain elusive.
Alternatively, fibrosis, a key clinical feature of POIKTMP, may also result from the failure of ECM-producing fibroblasts to undergo apoptosis after tissue remodeling. Resistance to apoptosis in idiopathic pulmonary fibroblast, alveolar macrophages, and impaired re-epithelization due to increased apoptosis of epithelial cells have been documented as the leading cause of collagen over deposition, further supporting this hypothesis (47)(48)(49)(50). Moreover, bioinformatics and experimental studies imply high FAM111B mRNA and protein expressions in epithelial cells/tissues of cancer and non-cancerous tissues (13,14,51). Thus, as described previously, it is conceivable that the low expression or rapid degradation of FAM111B caused by the FAM111B mutations could lead to increased epithelial apoptosis and, consequently, the resistance to apoptosis of adjoining fibroblasts of the tissues affected by POIKTMP.
Therefore, we proposed that the mutational dysfunction of FAM111B protein causes POIKTMP by downregulating FAM111B gene and protein expression that are resulting in inadequate DNA repair, genome instability, chronic inflammation, and aberrant apoptosis of the epithelial cells and fibroblasts of the tissues/organs affected by this disease (Figure 2).

ROLE OF FAM111B IN CANCERS
The overexpression and mutations of the FAM111B gene are associated with several types of cancers, including pancreatic cancer, which happens to be one of the most life-threatening types of cancer (11,12,15). In addition, the overexpression of FAM111B in lung adenocarcinoma (LUAD) patients correlated strongly with increased tumor progression and poor survival rate (13,14). There is, however, no evidence to support the degradation or clearance of endogenous FAM111B in cancers. Furthermore, multiple single-nucleotide polymorphisms (SNPs) associated with prostate cancer localize on chromosome 11q12, which houses the FAM111B and FAM111A genes (52). Given the possible involvement of FAM111B in DNA repair (5,10,29), the overexpression of the FAM111B gene can support cancer progression. One possible explanation could be that the overexpression of FAM111B results in the non-specific proteolytic degradation of other DNA-associated proteins such as histones and replication or transcription factors (e.g., RFC1 and RPB1) and cell-cycle-dependent proteins (e.g., p16). FAM111 B's activity switches on in normal states upon detecting DPCs, perhaps through p53 signaling. The activation of FAM111B will then proteolytically cleave the DPCs to repair the DNA. However, once the repair is complete FAM111B may undergo autocleavage to prevent itself from cleaving other proteins involved in DNA metabolism and the cell cycle (29). Therefore, the overexpression of FAM111B and the non-specific degradation of DNA-associated proteins may cause genomic instability and dysregulation of the cell cycle, eventually contributing to cancer predisposition, development, and progression ( Figure 2). Furthermore, to salvage genome stability and the apoptotic fate of cells, the error-prone translesion (TLS) DNA polymerases are mobilized to replace conventional high-fidelity DNA polymerases, which can overcome the DPC barrier to DNA replication (36, 53). This DNA damage tolerance mechanism and the uncontrolled degradation of tumor-suppressor proteins, p16, could explain why cancer cells can evade apoptosis. Another possible explanation is that FAM111B/FAM111A-repair machinery confers some advantage to cancer cells at later stages of cancer, thus supporting cancer progression. For instance, in cervical cancer patients, FAM111B, among other genes, is only overexpressed in later stages of cancer, and it is associated with distal cancer progression and metastasis (26,27,36). Therefore, it is plausible that FAM1111 B's upregulation is an adaptation strategy by cancer cells as most cancer drugs induce DNA damage by forming DPCs. In other words, FAM111B overexpression is an attempt to minimize genomic instability brought about by the formation of DPCs and conferring resistance to therapy.

DISCUSSIONS AND CONCLUSIONS
Based on the published literature, one can conclude that the molecular functions of FAM111B include the response to DNA damage resulting from DPCs, which can stall DNA replication and cause breaks in the DNA strands and genome instability. The DPCs are possible triggers for p53 activation, activating the downstream signaling proteins, eventually starting FAM111B and FAM111A. These two proteins may function individually or by forming a complex with other DNA repair proteins (CAPNS1-CAPN1, USP1, and FANCD2) to degrade DPCs to facilitate DNA repair and resumption of DNA replication ( Figure 1) (25). Furthermore, FAM111B may function with other DPC proteases, DNA repair, replication, and transcription proteins to restore genome stability, cell cycling, and turnover (25,26,36). If the repair is successful, FAM111B will relieve the cell cycle arrest by degrading p16 so that cell division can resume as normal. Also, FAM111B, as a possible regulated protease, should undergo autocleavage to prevent nonspecific cleavage of chromatin-associated proteins such as histones and transcription factors (37). If the repair is not successful, FAM111B, among other proteins, triggers the expression of BAG3 and BCL-2 genes to induce apoptosis of the cell (Figure 2) (25).
In the case of the mutated form of this protein, which is commonly a gain of function, the proteolytic activity of FAM111B may be amplified, resulting in the non-specific cleavage chromatin-associated proteins, among others crucial. Additionally, other essential functions of FAM111B, such as activating apoptosis, may be lost. Finally, since FAM111B is critical for degrading p16 for cell cycle progression, the mutations may result in abnormal degradation of p16 and thus cause abnormal cell division ( Figure 1B). As a result, FAM111B mutations tend to cause diseases such as POIKTMP and cancer FIGURE 2 | Proposed pathological processes involving FAM111B in cancer and POIKTMP. An increase in FAM111B expression possibly leads to an increase in proteolysis of DPCs and non-specific degradation of chromatin-associated proteins, which leads to genomic instability. Genomic instability is a significant characteristic of cancer. A decrease in FAM111B possibly leads to the reduction in apoptosis of fibroblasts, which causes an increase in fibrosis (Created with Biorender.com). through these mechanisms (Figure 2). More importantly, FAM111B seems more critical in some cell types than others. For example, the knockdown of FAM111B in Hela cells did not affect cell proliferation but did in lung adenocarcinoma cell lines. Thus, FAM111B mutations can affect certain cell types but spare the others (13,14).
Although the pathological knowledge effects of the mutant forms FAM111B are emerging, only a handful of studies have investigated the physiological function of FAM111B (which remains unclear) in healthy cells/tissues. Therefore, the proposed functions may lack accuracy since most reported studies were conducted using cancer cells. Therefore, future studies to establish the molecular function of this protein should be conducted in both healthy and disease-relevant models.
In conclusion, the human FAM111B protein is crucial to specific cellular processes essential for cell viability or fitness. Therefore, it is imperative to study the dysregulation of this protein in the context of fibrosing diseases like POIKTMP and cancers. Moreover, FAM111B represents a promising therapeutic target for both disorders.

AUTHOR CONTRIBUTIONS
MM: writing and visualization; FS: writing and visualization; CR: writing; AA: conceptualization, writing, editing, and supervision.


Validation of saline, PBS and a locally produced VTM at varying storage conditions to detect the SARS-CoV-2 virus by qRT-PCR

Coronavirus Disease-2019 tests require a Nasopharyngeal (NP) and/or Oropharyngeal (OP) specimen from the upper airway, from which virus RNA is extracted and detected through quantitative reverse transcription-Polymerase Chain Reaction (qRT-PCR). The viability of the virus is maintained after collection by storing the NP/OP swabs in Viral Transport Media (VTM). We evaluated the performance of four transport media: locally manufactured (“REVITAL”) Viral Transport Media (RVTM), Standard Universal Transport Media (SUTM), PBS and 0.9% (w/v) NaCl (normal saline). We used laboratory cultured virus to evaluate: i) viral recovery and maintaining integrity at different time periods and temperatures; ii) stability in yielding detectable RNA consistently for all time points and conditions; and iii) their overall accuracy. Four vials of SARS-CoV-2 cultured virus (2 high and 2 low concentration samples) and 1 negative control sample were prepared for each media type (SUTM, RVTM, PBS and normal saline) and stored at the following temperatures, -80°C, 4°C, room temperature (25°C) and 37°C for 7 days. Viral Ribonucleic acid (RNA) extractions and qRT-PCR were done on the following days after inoculation with the cultured virus, days 1, 2, 3, 4 and 7 to assess virus stability and viral recovery. CT values fell over time at room temperature, but normal saline, PBS, RVTM and SUTM all showed comparable performance in maintaining virus integrity and stability allowing for the detection of SARS-CoV-2 viral RNA. Overall, this study demonstrated that normal saline, PBS and the locally manufactured VTM can be used for COVID-19 sample collection and testing, thus expanding the range of SARS-CoV-2 viral collection media.

Kenya, like several countries during the COVID-19 pandemic, suffered from disruptions in the 80 supply of reagents required for diagnosis. It was therefore imperative to seek alternatives that 81 would cushion diagnostic centres from unforeseen shortages. Consequently, the present study 82 evaluated the performance of four transport media, i.e., a locally manufactured (i.e. "REVITAL") States). We first generated a10-fold dilution series (viral dilutions 1 to 10, S1 Table)   control) were prepared for each media type and stored at the following temperatures, -80°C, 4°C, 104 room temperature (25°C) and 37°C for 7 days (Fig 1), generating a total of 80 vials. Viral 105 Ribonucleic acid (RNA) extractions and qRT-PCR were done on the following days after 106 inoculation with the cultured virus, days 1, 2, 3, 4 and 7 to assess virus stability and viral recovery. QIAamp Mini Spin Columns. The spin columns were spun at 6,000 x g for 1 minute, the elute was 128 discarded and 500μl of buffer AW1 was added followed by centrifugation at 6,000 x g for 1 minute.
The flowthrough was discarded and 500μl of buffer AW2 was added followed by centrifugation 130 at 16,000 x g for 3 minutes. The elute was discarded and the spin columns allowed to air dry for 5 131 minutes before elution with 60μl buffer AVE.  presentations were produced in R version 3.6.1(13). First, we note that there were 128 negative samples defined as "undetermined" that were replaced with an arbitrary high Ct for negative (i.e., 152 37) to uncover the bias that might occur due to their exclusion during statistical analysis. Temporal

Comparisons between the 4 media types 187
For all temperature levels, media types and days the Ct values were consistently highest in the low 188 concentration samples (between Cts 25 and 28) and they were significantly higher than those for 189 high concentration samples (between Ct 18 and 23) (Tables 1 and 2, Fig 2). The Ct values varied 190 over time in the four temperature levels examined (Table 2). Ct values generally increased over 191 time (day) in the media stored at -80ºC and 4ºC, decreased and remained the same at room 192 temperature and 37ºC, respectively (Fig 2). The multiple linear regression analysis demonstrated 193 there were no statistically significant differences in Ct values across the media types (    qRT-PCR test is reached and taking into consideration the variability between qRT-PCR kits.

257
There were some contaminated negative control samples (Ct<36) in <2% of the samples examined.

258
The contamination may have occurred during extraction or the compilation of qRT-PCR 259 reagents(18) due to amplicon carry-over given the high number of SARS-CoV-2 amplicons being 260 generated in the lab. It may also be a reflection of the high signal-to-noise ratio at high Ct values. 261 We had mitigated against contamination by incorporating proper disinfection, and use of dedicated 262 working spaces ensuring minimal viral contamination (18). Samples paired with positive negative 263 controls were regarded as unreliable and were removed from further analyses.

264
These results based on laboratory cultured samples stored at a range of temperatures suggest that 265 samples swabbed directly from patients could be transported in the different media types examined 266 in this study, given the comparable ability to detect the viral RNA. However, we did not test 267 whether the different media types support viable culturable virus.

268
The cultured virus we used may be at higher concentration than samples taken in the field, and 269 storage conditions during transport may be more variable. However, we did not observe evidence 270 of any differences in RNA levels based on the PCR results by VTM, including when virus was 271 stored at room temperature.

272
Overall, this study demonstrated that normal saline, PBS and the locally manufactured VTM can 273 be used for COVID-19 sample collection and testing, thus expanding the range of SARS-CoV-2 274 viral collection media. 276 We thank Prof George Warimwe's group from KEMRI-Wellcome Trust Research Programme for 277 the cultured SARS-CoV-2 virus aliquot they kindly provided for use in this study. We thank the 278 KEMRI Director for permission to publish this work.


s2fieldsofstudy:
Biology
Materials Science

text:
Enhanced Eradication of Enterococcus faecalis Biofilms by Quaternized Chitosan-Coated Upconversion Nanoparticles for Photodynamic Therapy in Persistent Endodontic Infections

Due to the persistent presence of Enterococcus faecalis biofilms in apical root canals, persistent endodontic infections (PEIs) have always been an intractable disease to solve. The conventional root canal disinfectants (e.g., calcium hydroxide, chlorhexidine) are arduous to scavenge the stubborn infection. With the progress of nanomedicine in the biomedical field, antimicrobial photodynamic therapy (aPDT) is emerging as a prospective anti-infective therapy for PEIs. Herein, quaternized chitosan (QCh) modified upconversion nanoparticles (UCNP)@SiO2/methylene blue (MB) are developed with enhanced antibacterial/biofilm performance for aPDT in PEIs. QCh is coated on the UCNP@SiO2/MB by testing the changes in diameter, chemical functional group, and charge. Interestingly, QCh also increases the conversion efficiency of UCNP to generate more reactive oxygen species (ROS). Furthermore, the prepared UCNP@SiO2/MB@QCh exhibits highly effective antibacterial activity against free E. faecalis and related biofilm in vitro and extracted teeth. Importantly, the additional QCh with positive charges enhance UCNP@SiO2/MB@QCh contact with E. faecalis (negative charges) through electrostatic interaction. Then, UCNP@SiO2/MB@QCh could stick close to the E. faecalis and generate ROS under the irradiation by a 980 nm laser. The in vitro cellular test shows that UCNP@SiO2/MB@QCh has acceptable cytocompatibility. Thus, UCNP@SiO2/MB@QCh could offer a novel strategy for the potential aPDT clinical applications in the treatment of PEIs.

INTRODUCTION
Apical periodontitis (AP) is a destructive inflammatory disease of the apical periodontium (periodontal ligament, cementum, and bone), which resulted from persistent endodontic infections (PEIs) (Bordagaray et al., 2021). A new systematic review study from 2012 to 2020 has revealed that there is a notably increased prevalence of PEIs in adults compared with data from 2012 (6.3 vs. 5.4%) (Jakovljevic et al., 2020). Considering the stubbornly high prevalence, practical and effective treatment for PEIs is urgent (Wang et al., 2020a).
Enterococcus faecalis (E. faecalis) is a crucial etiologic pathogen of PEIs, and the pathogenicity of E. faecalis in biofilms is 100-1,000 times higher than in planktonic states (Bowen et al., 2018). However, relevant research reported that more than 40% of E. faecalis clinical isolates could form biofilms, so that AP becomes refractory (Feng et al., 2021;Ji et al., 2021). In the territory of EIs, the methods applied to eliminate bacteria from the root canal system mainly incorporate calcium hydroxide and antibiotics such as chlorhexidine. However, these medicaments are often associated with unsatisfied curative effects and inevitable adverse effects, such as discoloration of the tongue and teeth caused by chlorhexidine (Lei et al., 2016). Hence, developing a high-performance antibacterial biomaterial for eradicating E. faecalis biofilm is critical to the treatment of PEIs.
With the progress of nanomedicine in the biomedical field (Wang et al., 2021b;Qi et al., 2022;Yan et al., 2022), antimicrobial photodynamic therapy (aPDT) has attracted increasing attention as a promising means to scavenge pathogenic microorganisms GRAPHICAL ABSTRACT | Schematic diagram of the preparation of UCNP@SiO 2 /MB@QCh and the following antibacterial effect. from infectious lesions (de Annunzio et al., 2018;Zhang et al., 2020). Generally, the bactericidal mechanism of aPDT relies on the reactive oxygen species (ROS), which is produced by the photosensitizers (PSs) and oxygen under the irradiation of light with a particular wavelength (Hu et al., 2018;Zhao et al., 2021). Nevertheless, commonly clinically used PS, such as methylene blue (MB), have some deficiencies, including poor water-solubility, uncontrollable drug-release profile, poor target selectivity, and low extinction coefficient (Yin et al., 2015). In addition, their excitation light, such as ultraviolet light, causes tissue damage and visible light possesses inadequate tissue-penetration ability. To overcome the abovementioned limitations, lanthanide-doped upconversion nanoparticles (UCNPs) could serve as PSs or PS carriers owing to the merits of UCNPs (e.g., high photostability, low cytotoxicity, and sharp emission lines) (Lu et al., 2016;Lee et al., 2020). UCNPs could be able to absorb near-infrared (NIR) light and convert it into high-energy photons. The NIR light ranging from 700 to 1,100 nm had advantages of deeper tissue penetration and lower autofluorescence and could reduce side effects such as phototoxicity and photodamage. To enhance the efficiency of upconversion luminescence (UCL), a silica layer was applied to carry PSs on the surface of UCNPs, such as dense silica and mesoporous silica nanoparticles Bayir et al., 2018). For instance, Liu et al. verified the antibacterial effect of UCNP@SiO 2 /MB against Staphylococcus aureus and Escherichia coli. The death rates of these two bacteria were achieved at 37.56 ± 4.00% and 63.97 ± 5.26%, respectively . The antibacterial effect of UCNP@SiO 2 /MB was relatively low for Gram-negative bacteria, as the negatively charged nanoparticles were difficult to contact and interact with negatively charged bacteria rapidly and directly (Yue et al., 2018;Ahmed et al., 2020). Chitosan and its derivatives have positive charges. Chitosan has native characteristics, including biocompatibility and nontoxicity (Hao et al., , 2022Zheng et al., 2021). However, the relatively low solubility in physiological solutions restricts the use of chitosan. In contrast, the quaternized chitosan (QCh) has excellent water solubility. Meanwhile, it could enhance the conversion efficiency of UCNP. Hence, we chose QCh to modify UCNP, instead of chitosan. It is well-demonstrated that QCh with good biocompatibility and strong positive charges could identify, interact, and disturb the membrane of bacteria, which might increase the membrane permeability and lead them to death (Ao et al., 2020;Phuangkaew et al., 2022). Therefore, UCNP@SiO 2 /MB@QCh would offer a novel strategy to treat E. faecalis associated PEIs for potential clinical applications, which has not been reported.
In our study, we designed a novel triple-layered coreshell nanostructure UCNP@SiO 2 /MB@QCh to eradicate E. faecalis for the treatment of PEIs. The antibacterial effects of UCNP@SiO 2 /MB@QCh against free E. faecalis and related biofilms were demonstrated in vitro through aPDT. Besides, the model of E. faecalis biofilm on extracted teeth was established to investigate their antibacterial effects further. The antibacterial experiment results showed that the antibacterial performance of the developed nanomaterials holds great potential for the management of PEIs.
After reacting for 20 min, the temperature of the mixture solution was reduced to 25 • C. The mixture solution was divided and poured into two centrifuge tubes, and an equal amount of ethanol absolute was injected to static settlement for 30 min. Finally, the mixture was centrifuged and the sediment was dissolved into cyclohexane.

Synthesis of UCNP@SiO 2 /MB
To achieve a thin dense silica layer, UCNP@SiO 2 /MB was synthesized through a successful method . First, 40 ml cyclohexane and 2 ml igepal co-520(NP-5) were stirred for an hour. Then, 40 mg UCNPs were added to the flask and stirred violently for 3 h. Then, 280 µl ammonia solution and 900 µl methylene blue (4 mg/l) were injected dropwise, respectively. After mixing for 2 h, the combined solution of tetraethyl orthosilicate (TEOS) and cyclohexane was added slowly by a TJP-3A Syringe Pump Controller (LongerPump) for an hour. After 24 h, 10 ml formaldehyde aqueous solution was injected to mix for 30 min. Finally, the compound was centrifuged and the sediment was dissolved in double distilled water (DDW) using ultrasound.

Synthesis of UCNP@SiO 2 /MB@QCh
QCh was prepared by a reported procedure (You et al., 2016). First of all, the purified QCh was dissolved into DDW through ultrasound. Then, the liquid of QCh whose concentration was twice that of UCNP@SiO 2 /MB was added into the latter dropwise under ultrasound. The ultrasound procedure lasted for 30 min. After the abovementioned procedures, the mixture liquid was centrifuged for 10 min at 12,000 rpm. The products were washed with DDW twice and finally dispersed in DDW for future application.

Characterizations
Transmission electron microscopy (TEM) was obtained by Mic JEM-1200EX (Japan) to confirm the size and morphology. The power X-ray diffraction (XRD) pattern was recorded on a Rigaku Ultima IV with Cu-Kα radiation (λ = 0.15418). The morphology of samples was detected using SEM (VEGA3, TESCAN, Czech) operated at an acceleration voltage of 10 kV. A Nicolet iN10 Fourier transform-infrared (FTIR) spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) was utilized to measure the FTIR spectra of the samples over the range of 500-4,000 cm −1 at a scanning resolution of 2 cm −1 during 32 scans. The luminescence intensity was recorded on an FLS980 fluorescence spectrometer (Edinburgh Instruments Ltd., UK) under 980 nm laser excitation at 1.5 W/cm 2 with a NIR laser device. The UV-Vis spectrum was collected on UV-Vis Spectrophotometer (UV-8000, Shanghai). The zeta potential (Z) and diameter distribution (DLS) of products was detected by dynamic light scattering on a Zetasizer Nano ZSE (UK). The 3D-images of bacteria biofilms were carried out on a confocal laser scanning microscope (CLSM, Leica TCS SP8, Germany).

Detection of Reactive Oxygen Species (ROS)
1,3-diphenylisobenzofuran (DPBF) was adopted to detect the generation of ROS from MB, indicating the PDT effect of nanoparticles. It is well known that DPBF can especially react with ROS, resulting in a decreased absorption intensity at 410 nm (Zhang et al., 2018b;Sun et al., 2020). The greater the reduction in absorption, the more ROS is generated. In brief, DPBF was dispersed in acetonitrile. MB, UCNP@SiO 2 , UCNP@SiO 2 /MB, and UCNP@SiO 2 /MB@QCh were prepared in ethanol absolute. Then, 20 µl DPBF (4 mg/ml) was added into 1.98 ml ethanol absolute containing the abovementioned materials, respectively. Moreover, 20 µl DPBF was resolved into ethanol absolute to act as the control group. The solution was irradiated by a 980 nm laser (1.5 W/cm 2 ), and the absorbance of DPBF at 410 nm was recorded every 5 min in darkness.

Cytotoxicity Assay
CCK8 (Cell counting kit-8, Meilunbio, Dalian) was applied for validating the cellular viability of UCNP@SiO 2 /MB@QCh. At first, the nanoparticles were dispersed in a DMEM culture medium at the concentration of 25, 50, 125, and 250 µg/ml. Meanwhile, mouse fibroblast L929 cells were seeded in 96-well plates at a density of 6 × 10 3 cells per well for 2 days at 37 • C . The control group contained 100 ml DMEM without UCNP@SiO 2 /MB@QCh. Then, 50 ml UCNP@SiO 2 /MB@QCh and 50 µl DMEM were added to cell plates, which means the ultimate concentrations of materials were 12.5, 25, 62.5, and 125 µg/ml. Then, the particles were incubated for 12, 24, and 36 h. After performing the abovementioned procedures, the residual liquid was removed from the wells and 100 µl fresh medium with 10 µl CCK-8 reagent solution was added to each well of the plate. Finally, OD450 was measured using a microplate reader. The relative cell viability was calculated by the following formula (Ji et al., 2021): In vitro Antibacterial Tests E. faecalis ATCC 29212 was selected as the standard species of Gram-positive bacteria. E. faecalis strain was incubated in a brain heart infusion (BHI) medium, resulting in a density of 1 × 10 7 colony-forming units (CFU) per ml. To avoid the heating effect of 980 nm laser irradiation, every cell of the 96-well plates containing 200 µl BHI with E. faecalis (1×10 7 CFU/ml) was explored with NIR laser (1.5 W/cm 2 ) for 10, 20, and 30 min, respectively. Furthermore, each well was irradiated for 1 min at an interval of 2 min (Zhang et al., 2018a;.
Then, 20 µl suspension was taken out from each well, diluted with sterilized PBS, dropped on BHI agar plates, and incubated at 37 • C for 24 h. The bacteria colony numbers of each group were calculated.
To select the appropriate irradiation time, 1.5 ml UCNP@SiO 2 /MB@QCh (2 mg/ml) and 1.5 ml bacterial solutions (1 × 10 7 CFU/mL) were mixed and co-cultured for 1 h in a shaking incubator. Then, 200 µl suspension was added to a 96-well plate and irradiated by a 980-nm laser for 10, 20, and 30 min, respectively. 1.5 ml bacterial suspension with 1.5 ml BHI medium was set as the control group without NIR irradiation. However, the procedure was the same as that mentioned in the above experiment.
To investigate the antibacterial effect of UCNP@SiO 2 /MB and UCNP@SiO 2 /MB@QCh, 1 ml bacterial suspension was added to 1 ml of the above materials at varying concentrations (25, 50, 125, and 250 µg/ml), respectively. Then, the mixture was incubated in a shaking incubator for an hour (Li et al., 2017). Next, 200 µl mixture suspension was illuminated by a 980 nm laser for 10 min . The control group included the solution of 1 ml bacterial strain and 1 ml BHI medium. The steps that were followed were the same as before. Finally, the survival rates of E. faecalis were figured out by the following equation: The relative survival rates = N Text /N Control ×100% Where N Text means the colony number of the experimental group, and N Control means the colony number of the control group .

Biofilm Inhibition Tests
First, the circle microscope cover glasses were placed in 75% alcohol for ultrasonic treatment for 30 min and then soaked for 8 h for disinfection. The drying surface liquid and the glasses were placed on a 12-well plate. Of note, 1 ml of E. faecalis suspension was added to each well and mixed properly, and the plate was placed in the incubator for 2 days. Then, the original medium was taken out. Meanwhile, 1 ml of fresh BHI medium was added to each well. In the control group, 1 ml of medium was added again, and in the experimental group, 1 ml UCNP@SiO 2 /MB@QCh (250 µg/ml) and 1 ml of CHX (2%) were added for co-culture for 1 h. Next, the wells with UCNP@SiO 2 /MB@QCh were exploded to 980 nm NIR irradiation for 10 min. Then, the liquid from each well was removed. Then, 2.25 µl PI and STYO9 were dissolved in 1.5 ml sterile DDW. Every cell-containing glass was injected with 200 µl of the mixture of PI and STYO9, and stained for 15 min. The solution was taken out and each glass was washed with PBS 3 times. Finally, fluorescence images from the stained bacteria were observed under a confocal laser scanning microscope (CLSM, Leica TCS SP8, Germany). Bacterial cell viability was determined by using propidium iodide (PI) and STYO9 staining dye (Zhu et al., 2020). Live bacteria were stained into green fluorescence and dead bacteria were stained into red fluorescence.
To observe the morphology of E. faecalis, the dentin plates from the third molar were adopted to culture E. faecalis strain.
The plates were added to a 12-well plate and immerged in a 2ml bacterial suspension. The culture lasted for 7 days, and the fresh culture BHI medium was substituted every 2 days. Then, 2 ml BHI medium with UCNP@SiO 2 /MB@QCh (125 µg/ml) was added into three wells, while the control group was added in 2 ml BHI. After 1 h, the experimental group was exploded to 980 nm NIR irradiation for 10 min. After glutaraldehyde fixation and alcohol gradient dilution, the samples were tested by SEM.

Statistical Analysis
All data were shown as mean ± standard deviation (SD). All statistical analyses were evaluated using Graphpad Prism 8 or Origin 9. One-way analysis of variance (ANOVA) with Tukey's test was used to determine the differences between the groups. A value of p < 0.05 was considered to be statistically significant.

Physicochemical Characterization of UCNP@SiO 2 /MB@QCh
UCNP@SiO 2 /MB and QCh were prepared by reported procedures You et al., 2016). Then, UCNP@SiO 2 /MB@QCh was synthesized utilizing their electrostatic interaction, forming core-shell structured lanthanide-doped upconversion nanoparticles (Yin et al., 2016). To validate the core-shell structure of UCNP@SiO 2 /MB@QCh, TEM was employed to detect their structure at the nanoscale level. As shown in Figure 1A, the nanosized UCNP (∼98 nm) presented the form of a pure hexagonal phase and dispersed homogeneously. Native UCNP showed low fluorescence resonance energy transfer (FRET) efficiency and considerable background, which may reduce the assay sensitivities (Li et al., 2015). To enhance the efficiency of FRET, as shown in Figure 1B, the dense silica layer with a thickness of 8 nm was smoothly wrapped around the UCNPs. After incorporation with QCh, the surface of UCNP@SiO 2 /MB@QCh became rough and its size increased to around 109 nm ( Figure 1C). Besides, a double-layer around the core was identified, that is, the layer next to the core was the silica layer and the outermost layer was QCh (Lv et al., 2017). Furthermore, the particle size of UCNP@SiO 2 /MB@QCh in the aqueous solution detected by DLS was 187 nm, while the particle size of UCNP@SiO 2 was 137 nm (Figure 1D). It may be concluded that the conjunction of QCh to UCNP@SiO 2 was successfully achieved by the comparison of diameters between UCNP@SiO 2 and UCNP@SiO 2 /MB@QCh. It was reported that when the nanomaterial size is smaller than 500 nm, they could better penetrate and destroy bacterial biofilms (Wu et al., 2021). The size of the prepared UCNP@SiO 2 /MB@QCh was much smaller than 500 nm, which indicates that it might enter inside bacterial films and then eradicate pathogenic bacteria.
It has been reported that nanoparticles with positive charges could interact and disturb the membrane of bacteria, which might increase the membrane permeability and lead them to death (Bing et al., 2016). Hence, we tested the charges of UCNP@SiO 2 , UCNP@SiO 2 /MB, and UCNP@SiO 2 /MB@QCh. As shown in Figure 2A, it was found that the zeta potential of UCNP@SiO 2 was −30.8 mV, while that of UCNP@SiO 2 /MB was −20.2 mV. Conversely, the zeta potential of UCNP@SiO 2 /MB@QCh changed to 28.9 mV owing to the modification of positively charged QCh, which also reveals the successful construction of UCNP@SiO 2 /MB@QCh (Qiao et al., 2020). Choudhary and co-workers reported that nanofluids were regarded as moderately stable when their zeta potential was near ±30 mV (Choudhary et al., 2017).
Additionally, XRD patterns of UCNP and UCNP@SiO 2 /MB@QCh indicated that the prepared nanoparticles were in the form of a hexagonal phase ( Figure 2B). The chemical feature of QCh, UCNP@SiO 2 /MB, and UCNP@SiO 2 /MB@QCh was measured by FTIR analysis (Figures 2C,D). After salinization, UCNP@SiO 2 /MB showed two strong absorption bands at 1,066 and 800 cm −1 due to asymmetric, symmetric stretching vibrations and bending vibrations of the Si-O-Si bond . This provided an elucidation for the successful synthesis of UCNP@SiO 2 /MB. In addition, the special sharp peak of QCh was observed at 1,479 cm −1 , which corresponded to the methyl groups of ammonium (Song et al., 2010;You et al., 2016). It was also obtained at 1,472 cm −1 in UCNP@SiO 2 /MB@QCh. Meanwhile, the peak of C=O in QCh appeared at 1,656 cm −1 , which was detected at 1,650 cm −1 on the latter (Niamlang et al., 2017). These results imply that QCh was successfully coated onto UCNP@SiO 2 /MB. Furthermore, as shown in Figure 3A, the UV-Vis spectra of MB, UCNPs@SiO 2 , and UCNPs@SiO 2 /MB indicated that MB was successfully loaded into the SiO 2 layer (Yue et al., 2018).

Luminescence Ability and ROS Formation Induced by UCNP@SiO 2 /MB@QCh
To confirm the luminescence ability of the samples, we tested the relative luminescence intensity of UCNPs@SiO 2 -based nanoparticles. Findings revealed that the strong absorption peak of MB was 654 nm and the emission wavelength of UCNP was 650 nm, which matched well (Yin et al., 2014). Therefore, the FRET between MB and UCNPs may exist with high efficiency to further generate increased ROS to eradicate pathogenic bacteria. Figure 3B shows that the luminescence intensity at 650 nm of UCNP@SiO 2 /MB was reduced by 64% compared with UCNPs@SiO 2 . UCNP@SiO 2 /MB@QCh decreased by 88%. Interestingly, QCh may contribute to enhancing the FRET efficiency of the nanoparticles.
The antimicrobial application of UCNP@SiO 2 /MB@QCh mainly depends on its ability to produce ROS species, especially singlet oxygen ( 1 O 2 ) (Wang et al., 2020b;Zhu et al., 2022). Due to the greater penetration of NIR laser compared with visible light, UCNP@SiO 2 /MB@QCh could be applied for antimicrobial activity in deeper tissues, such as apical periodontium. In our work, UCNP could activate MB to product 1 O 2 to react with DPBF. This could generate uncolored 1,2dibenzoylbenzene (DBB) as the product (Qi et al., 2019;Zhao et al., 2020). Subsequently, the absorption value of DPBF at 410 nm would decrease with the generation of DBB. As shown in Figure 3C, the degradation rate of MB or UCNP@SiO 2 alone dropped a little after irradiation of 980 nm NIR, while that of DPBF did not change. However, the degradation rate of UCNP@SiO 2 /MB@QCh significantly decreased by 82% at 50 min, while that of UCNP@SiO 2 /MB reduced by 38%. Besides, the degradation rates decreased gradually with the extension of NIR time. Therefore, we may conclude that compared with UCNP@SiO 2 /MB, UCNP@SiO 2 /MB@QCh produced a significant amount of 1 O 2 and could possess a higher photodynamic ability, which was supposed to play an important role to eliminate bacteria. It has been reported that QCh may increase the conversion efficiency of UCNP to generate more ROS (Liao et al., 2019). Another striking example reported by Hu and coworkers showed the crucial effect of ROS to irreversibly destroy the membrane structure of bacteria, thus leading to the death of bacteria .

Cytocompatibility and Antibacterial Assessment of UCNP@SiO 2 /MB@QCh
The potential cytotoxicity of nanoparticles is a crucial indicator in biomedical applications. Thus, the cytotoxicity assay was performed on L929 fibroblasts in our work Wu et al., 2019). As demonstrated in the result of the CCK-8 assay (Figure 4A), the cell viabilities of L929 fibroblasts with UCNP@SiO 2 /MB@QCh in different co-culture times were all above 90%, indicating that the toxicity of nanoparticles toward cells might not significantly increase with time. In addition, after incubation with different concentrations of UCNP@SiO 2 /MB@QCh, the cell viabilities of L929 fibroblasts were nearly 90%, which suggests excellent cytocompatibility of the developed nanoparticles.
Each well of E. faecalis was irradiated for 1 min and at an interval of 2 min to avoid the heating effect of NIR light. As shown in Figure 4B, the vitality of E. faecalis significantly decreased when the irradiation time was 30 min, while their changes were negligible at 10 and 20 min. It could be considered that NIR irradiation at 10 and 20 min could not reduce the vitality of E. faecalis. Furthermore, to identify the proper irradiation time, the mixture of E. faecalis and UCNP@SiO 2 /MB@QCh was explored using a 980 nm laser for 10, 20, and 30 min, respectively. The vitalities of E. faecalis were significantly declined when irradiated for more than 10 min. Therefore, the irradiation time of 10 min was selected in the following experiment ( Figure 4C).
To investigate the antibacterial effect of UCNP@SiO 2 /MB@QCh, E. faecalis was incubated with the nanomaterials for 1 h and irradiated by 980 nm NIR for 10 min (Lv et al., 2022). We found that UCNP@SiO 2 /MB@QCh displayed a better antimicrobial effect than UCNP@SiO 2 /MB with or without NIR laser (Figures 5A,B). As shown in  Figures 5A,B, the survival rates subsequently decreased with the increasing concentration of UCNP@SiO 2 /MB@QCh. When the concentration arrived at 250 µg/ml, the survival rate of E. faecalis reduced to 0.6% without NIR irradiation. Meanwhile, it decreased to 0.2% after NIR irradiation, revealing that ROS could further lead E. faecalis to death. The reason may be that UCNPs were excited by NIR irradiation, which made MB generate ROS to kill bacteria (Wang et al., 2021a;Wu et al., 2022). Giannelli and co-workers confirmed that MB can produce single oxygen under specific conditions, which may generate hydroxyl radicals(OH-) and other kinds of ROS to remove bacteria (Giannelli and Bani, 2018). Due to the coating of QCh, UCNP@SiO 2 /MB@QCh exhibited positive charges.
Hence, it may identify, adhere to, and destroy E. faecalis more efficiently. Cheah et al. reported that chitosan and its derivatives disrupt bacteria integrity and the intracellular components are leaked (Cheah et al., 2019). Therefore, UCNP@SiO 2 /MB@QCh obtained a better antibacterial effect than UCNP@SiO 2 /MB. UCNP@SiO 2 /MB@QCh performed an excellent inhibiting effect against E. faecalis, showing a promising application prospect.
Biofilm Inhibition Evaluation of UCNP@SiO 2 /MB@QCh E. faecalis biofilm is a key primary cause of PEIs (El karim et al., 2007;Keskin et al., 2017). To further verify the antibiofilm properties of UCNP@SiO 2 /MB@QCh, we conducted antibacterial experiments on the biofilm of E. faecalis. As shown in Figure 6, a uniform green E. faecalis biofilm was detected in the control group, which demonstrated that a uniform biofilm model was established (Qi et al., 2021). In the experimental group, after being treated with UCNP@SiO 2 /MB@QCh, the area of green fluorescence narrowed down, and the red fluorescence showed higher intensity and wider range, which indicates that its biofilm structure was loose, and its biofilm thickness decreased. Importantly, the anti-biofilm ability of UCNP@SiO 2 /MB@QCh is comparable to commercial chlorhexidine.
As shown in Figure 7, SEM was employed to detect the effect of UCNP@SiO 2 /MB@QCh on E. faecalis biofilm on the root canal surface. It was found that the amount of E. faecalis in the UCNP@SiO 2 /MB@QCh group was greatly decreased compared with that of the control. The E. faecalis biofilm in the UCNP@SiO 2 /MB@QCh group was destroyed, and its bacteria were sparsely dispersed. QCh could adsorb bacteria and then destroy their cell membranes, resulting in the efflux of DNA and RNA from the cytoplasm, thus achieving the effect of killing bacteria (Abd El-Hack et al., 2020). Combined with the action of ROS, UCNP@SiO 2 /MB@QCh achieved an antibacterial double impact.
for the management of PEIs. The size of UCNP@SiO 2 /MB@QCh (187 nm) was significantly larger than that of UCNP@SiO 2 (137 nm) due to the coating of QCh. In the course of aPDT treatment, more than 99% of E. faecalis were eliminated by the UCNP@SiO 2 /MB@QCh. Furthermore, accompanied by the modification of positive charge QCh, the germicidal efficiency  was remarkably higher than that of UCNP@SiO 2 /MB. Such excellent antibacterial performance benefited from the close attachment with bacteria caused by QCh. Also, as a proverbial biocompatible and non-toxic material and widely used in biological fields, the addition of QCh greatly reduced the toxicity of aPDT photosensitizer and improved its biosafety. Thus, UCNP@SiO 2 /MB@QCh provides a novel strategy to eradicate PEI-associated bacteria for potential practical applications.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.

AUTHOR CONTRIBUTIONS
BZ and XL carried out the experiments, performed data analysis, and wrote the manuscript. QX designed the experiments, analyzed the results, and wrote the manuscript. PG analyzed the results and wrote the manuscript. QZ designed the experiments, revised the manuscript, and did project administration. All authors read and approved the final manuscript.